1. Field of the Invention
This invention relates to a co-expression system which comprises a gene coding for protein disulfide isomerase (PDI), a gene coding for an yeast receptor protein ERD2 or analog thereof and a foreign gene coding for a useful polypeptide and to a process for the production of said polypeptide using said system. PDI is an enzyme which enhances formation of the higher-order structure of polypeptides through its function of catalyzing the exchange reaction of a disulfide bond(s) in the polypeptides.
2. Prior Art
Studies on the in vitro refolding of denatured proteins have revealed the presence of both isomerization reactions of a disulfide bond and of a proline peptide as factors for determining a folding rate of polypeptides (Freedman, Cell, vol.57, pp.1069-1072, 1989; Fisher and Schmid, Biochemistry, vol.29, pp.2205-2212, 1990). As enzymes which catalyze these slow reactions during the polypeptide folding, peptidyl prolyl cis-trans isomerase (PPI) has been found in the latter case, and protein disulfide isomerase (PDI) and thioredoxin in the former case. According to in vitro experiments, these enzymes accelerate a refolding rate of denatured proteins, thus indicating a possibility of applying them to the in vitro refolding of inactive proteins produced by genetic engineering techniques (Schein, Bio/Technology, vol.7, pp.1141-1148, 1989; J. Udaka, Nippon Nogei Kagaku Kaishi, vol. 64, pp. 1035-1038, 1990).
Since PDI is soluble in water and can be isolated relatively easily from the liver of mammals, its properties as a catalyst have been studied in detail. PDI catalyzes the exchange reaction between thiol/disulfide bonds and is capable of undergoing formation, isomerization or reduction of the disulfide bond in protein substrates (Freedman, Cell, vol.57, pp.1069-1072, 1989). It is known that, in vitro, PDI enhances the formation or exchange reaction of a disulfide linkage(s) in molecules of a single domain protein such as RNase and of a multiple domain protein such as serum albumin, or enhances the formation of an intermolecular disulfide bond(s) in a protein having a subunit structure such as immunoglobulin, procollagen or the like (Freedman, Nature, vol.329, p. 196, 1987).
PDI from Mammals exists usually as a homodimer of the polypeptide having a molecular weight of about 57,000 and shows a highly acidic pI value (4.2 to 4.3).
The PDI gene from rat liver has been isolated. The amino acid sequence deduced from the DNA sequence of the PDI gene indicated that PDI has an intramolecular duplicate structure consisting of two homologous units. One of these two homologous units has a homology to the amino acid sequence of thioredoxin, indicating that its active site has an amino acid sequence similar to that of thioredoxin (Edman et al., Nature, vol.317, pp.267-270, 1985). Thioredoxin enhances the reduction of a disulfide bond in insulin and the exchange reaction of a disulfide bond in RNase in vivo, which indicate that thioredoxin plays a similar role to PDI in the in vivo folding process of proteins (Pigict and Schuster, Proc. Natl. Acad. Sci., U.S.A., vol.83, pp.7643-7647, 1986).
Although the amount of PDI present in a mammalian living body differs depending on the type of tissues and the differentiation stage of cells, such a difference is probably attributed to the existence of certain secretory proteins. In addition, PDI is localized abundantly in the endoplasmic reticulum through which a protein destined for secretion is known to pass. On the basis of these facts, it is assumed that PDI catalyzes or accelerates the formation of a disulfide bond(s) in secretory proteins newly synthesized within cells. Such an assumption is supported by the results of a study on the biosynthesis of .gamma.-gliadin in a cell-free protein synthesis system, that the formation of a disulfide bond in conjunction with the translation of .gamma.-gliadin hardly occurs when an endoplasmic reticulum fraction from which PDI was washed out in advance is used, while the disulfide bond formation is restored by the addition of PDI (Bulleid and Freedman, Nature, vol.335, pp.649-651, 1988).
In addition to the disulfide bond formation, PDI functions in other post-translational modifications of proteins. For example, the multifunctional property of PDI in connection with the protein modifications has been suggested on the basis of its homology to a catalytic unit, .beta.-subunit, of prolyl-4-hydroxylase which catalyzes hydroxylation of proline residues in collagen, to a glycosylation site binding protein that recognizes a signal sequence Asn-X-Ser/Thr of a peptide to which a sugar chain is bound during N-glycosylation process of synthetic protein (Pihlajaniemi et al., EMBO J., vol.6, pp.643-649, 1987; Geetha-Habib et al., Cell, vol.54, pp.1053-1060, 1988), to a thyroid hormone binding protein (triiodo-L-thyronine binding protein; Cheng et al., J. Biol. Chem., vol.262, pp.11221-11227, 1987), etc. In addition to these facts, some molecular species having partly homologous amino acid sequences to PDI have been found though they are different from the PDI. For example, certain gonadotropic hormones such as follitropin and lutropin contain amino acid sequences homologous to an amino acid sequence which is regarded as an active site of PDI, and these hormones catalyze the isomerization of a disulfide bond (Boniface et al., Science, vol.247, pp.61-64, 1990). Also, phospholipase C, an enzyme which hydrolyzes phosphatidylinositol-4,5-bisphosphate into 1,2-diacyl glycerol and inositol-1,4,5-triphosphate, has a domain homologous to PDI in its molecule (Bennett et al., Nature, vol.334, pp.268-270, 1988). In consequence, PDI and PDI-like molecules seem to have many functions in a markedly wide range of vital phenomena, both intracellularly and extracellularly.
Although the PDI has extensive functions as described above, a main effect of PDI is to form a protein (or a protein complex) having a natural higher-order structure by catalyzing the isomerization of an intramolecular or intermolecular disulfide bond(s). In many cases, however, an almost stoichiometric amount of PDI is required to attain an optimum reaction rate. It is expected therefore that an intramolecular or intermolecular isomerization rate of a disulfide bond will be slow when a disulfide isomerase has a low activity, and such a slow reaction rate will entail a low formation efficiency of a protein having a suitable disulfide bond(s). It is thought that such a low disulfide isomerase activity is one causes of the formation of insoluble molecular aggregates of various eukaryote-originated proteins (especially secretory proteins) in Escherichia coli. Although E. coli contains thioredoxin which is superior to PDI in terms of the activity as a disulfide reductase, the isomerase activity of thioredoxin is low. On the contrary, since an intramolecular disulfide bond(s) can often be found in secretory proteins, it is thought that a disulfide bond activity resulted from disulfide isomerization is high in cells or tissues which have a high secretion ability. This was indicated strongly by a comparative study on the relative PDI mRNA contents in various rat tissues, in which the contents in organs were found to be liver&gt;pancreas, kidney&gt;lung&gt;spermary, spleen&gt;heart&gt;brain in order (Edman et al., Nature, vol.314, pp.267-270, 1985).
Since polypeptides are synthesized in the cytoplasm which has reducing environment in the cells, a disulfide bond which is necessary for the proper folding of a polypeptide will not be formed efficiently in the reducing condition. Such a condition is generated for example in prokaryotic cells which have no compartments. Taking this into consideration, prokaryotic cells and eukaryotic cells may be different from each other in terms of factors concerning the formation of a disulfide bond and of conditions which enable its formation. When useful proteins (most of them are secretory proteins) are produced by recombinant DNA techniques, it is necessary to form a disulfide bond under certain conditions which are suitable for each protein to be produced. To accomplish such conditions, a host cell should have a suitable compartment and a large amount of a disulfide-forming enzyme (i.e., disulfide isomerase) which is localized in the compartment.
In eukaryotes, secretory proteins are transported outside the cell through the endoplasmic reticulum, Golgi body and secretory granules, and such a secretion process is regarded as a passive flow which is called "bulk flow". On the other hand, proteins localizing in the cavity of endoplasmic reticulum were initially thought to stay therein via such a process that the corresponding proteins synthesized were incorporated into the endoplasmic reticulum in the similar manner to the secretory protein and then transferred along the "bulk flow", but sent back again from Golgi body to the endoplasmic reticulum by a certain mechanism. Thereafter, primary structures of various proteins localizing in the mammalian endoplasmic reticulum have been determined. From some of these proteins, such as a protein disulfide isomerase (PDI), a glucose-regulated protein 78 (grp78, the same as Bip which is an immunoglobulin heavy chain binding protein) and a glucose-regulated protein 94 (grp94), a common C-terminal sequence "KDEL" ("HDEL" in the case of yeast) consisting of 4 amino acid residues was found. In addition, it was suggested that, in yeast cells, this sequence acts as a signal for allowing proteins to localize in the endoplasmic reticulum, because a mutant protein of Grp78 lacking in KDEL sequence is secreted extracellularly and because lysozyme, in spite of its secretory nature, can stay in the endoplasmic reticulum when the "HDEL" sequence is bound to its C-terminus (Munro, S. and Pelham, H. R. B., Cell, vol.48, p.899, 1987; Pelham, H. R. B., Hardwick, K. G. and Lewis, M. J., EMBO J., vol.7, p.1757, 1988). In consequence, it was considered that an endoplasmic reticulum or Golgi body contains receptor molecules specific for the "KDEL" or "HDEL" sequence, and that the receptor controls localization of a protein having such a sequence on its C-terminus.
Thereafter, a receptor for the signal "HDEL" was identified in yeast by the analysis of a yeast mutant erd2 in which a protein having "HDEL" sequence does not stay in the endoplasmic reticulum but transfers along its secretion process, and the receptor for the signal KDEL was also identified in mammals by the analysis in which anti-idiotype antibodies specific for "KDEL" sequence were used (Semenza, J. C., Hardwick, K. G., Dean, N. and Pelham, H. R. B. , Cell, vol. 61, p. 1349, 1990; Vaux, D., Tooze, J. and Fuller, S., Nature, vol.345, p.495, 1990). Gene structure of the yeast "HDEL" receptor has been revealed from which its primary amino acid sequence was deduced, with an estimated molecular weight of 26 kd. On the other hand, the mammalian "KDEL" receptor identified by the use of anti-idiotype antibodies has been reported to have a molecular weight of 72 kd, thus indicating that the mammalian receptor is probably different from the above yeast receptor. Thereafter, a gene coding for a protein homologous to the yeast "HDEL" receptor has been cloned in mammals by cross-hybridization (Lewis, M. J. and Pelham, H. R. B., Nature, vol.348, p.162, 1990). However, it is not clear whether the two different signal receptors function with mutual relationship or independently in mammals.
In addition to the "KDEL" and "HDEL" sequences, other homologous sequences such as "DDEL", "ADEL", "SDEL", "RDEL", "KEEL", "QEDL", "HIEL", "HTEL" and "KQDL" are known as signals for staying in endoplasmic reticulum, and polypeptides having these sequences are considered to stay in endoplasmic reticulum by associating with the aforementioned receptor molecules (Pelham, H. R. B., TIBS, vol.15, p.483, 1990).
However, nothing is in practice known about an in vivo system in which PDI is contained in a large quantity in a suitable compartment in the coexistence of a useful target protein, the PDI being capable of acting on the protein. Moreover, in spite of the applicability of PDI to the in vitro refolding of denatured proteins and to the improved productivity of secretory proteins in cells, this enzyme has been prepared only by direct purification from the internal organs. In addition, there are no reports on the interspecific expression of PDI, and on the establishment of a process for its production by means of genetic engineering or a process in which the productivity of a useful polypeptide is improved by the combination of the PDI gene with a gene coding for the polypeptide.